Method of extracting adrenal cortical hormones and the product of such method



Patented Sept. 8, 19.36

METHOD OF EXTRACTING ADRENAL'COB- TICAL HORMONES AND THE PRODUCT OF SUCH METHOD George F. Cartland and Marvin H. Kuizenga,

Kalamazoo, Mich.

No Drawing. Application January 10; 1935, Serial No. 1,210

3 Claims. (Cl. l67--'-77) This invention relates to the isolation from the high purity and is free from epinephrin and other adrenal glands of cattle or other mammals of objectionable amino compounds occurring naturbiologically active extracts containing the adrenal ally in mammalian adrenal glands.

. cortical hormone which is essential to life. Other objects and advantages will appear from 5 We have found that by following out the steps the description to" follow. The invention is 5 of our invention it is possible to produce from pointed out in the claims. mammalian adrenal glands an extract of such In carrying out our process, we use whole fresh adrenal cortical hormones which is of very high adrenal glands of slaughter-house animals which efiicacy in the treatment of deficiencies in such glands are frozen immediately after their re- ,lO hormones and that in following out the steps of moval at the slaughter-house and are kept froour invention it is possible to produce such an zen until desired for use. These glands from e extract that is of extreme purity and from which slaughte -house a p d in S d ca bon ditoxic and other objectionable impurities are sub- Oxide a d w n re d t the 'y are stantially absent. finely ground or chopped in a chopper such as a The objects of this invention are: food chopper and the finely ground or frozen 15 First, the provision of a new and improved glands are immediately mixed in acetone which method of producing such extract containing is saturated with carbon dioxide and in which adrenal orti al hor one, pieces of solid carbon dioxide are placed. Fol- Second, the provision of such a method of prolowing t Procedure keeps the glands in a 11'0- 29 ducing said extract which is simple and inexpen- Zen condition which Serves the p pose of presive and because of the nature of the various Serving h m and main ains them in this condisteps involved is well adapted to commercial protion until the acetone has had an Opportunity t0 duction of said extract from mammalian adrenal penetrate the tissue preserve the m In glands on a manufacturing scale in a, form suitaddition 130 this the carbon dioxide serves to 5 able for therapeutic use. acidify the mixture while keeping it cold during Third, to produce such a method that ii ithe dehydration and extraction of the fresh tissue nates the epinephrin and other amin o and maintains the mixture in an atmosphere of pounds, which constitute some of the most troucarbon dioxide excluding blesome impurities in the extract of adrenal cor- It Will be appreciated y ho e Sk lled in the tical hormone, from the final product, making a that instead of acetone, ethyl o p pyl alco- 30 possible the extraction from whole mammalian hcl could be emp y d or at a y solvent apa le adrenal glands without the necessity of dissecting of extracting the d e cort hor o es. the suprarenal cortex away from the medullary which solvent s a o wa ub e. ou d be tissue of the gland, which step is commercially p yprohibitive. In carrying out theinvention, we specifically 35 Fourth, to provide such a method which elimitake 28 kilos of the fresh ad lands and nates the heretofore involved, expensive and time extract them with 70 liters of aeetone P- consuming manipulations which are not suited p a y t e days a r m t p rature after to large scale commercial production and which the refrigerating ff of the carbon dioxide has 40 in many cases result in the loss or destruction of abated with mechanical agitation a f equent in- 40 a great part or all of the adrenal cortical hortel'vels- The Supernatant acetone 18 eip e ed ofl mone. and the tissue residue is extracted a second time Fifth, to produce such a method that easily with 56 liters of 80% acetone. The second aceand simply eliminates objectionable impurities. tone extract is sip Off and the tissue residue Sixth, to produce sucha method that employs is separated from the remaining solvent in a 45 as an extracting solvent for the adrenal cortical filter press. hormone ethylene dichloride. As the next step, we concentrate the combined Seventh, to provide such a method ln'which the acetone extracts in vacuo to remove the acetone whole mammalian adrenal glands are extracted and leave an aqueous residue which in the spein a solvent in the presence of solid carbon dicific instance has a, volume of approximately 28 5o oxide to preserve the glands during the extracliters. We then extract this aqueous residue tion and to exclude air from the glands during which contains the adrenal cortical hormones the first extraction step. with petroleum ether. This extraction is done Eighth, to provide a new and improved extract twice using seven liters for each extraction and of adrenal cortical hormone which extract is of the petroleum ether removes large quantities of fatty material but leaves practically all of the adrenal cortical hormones behind'in the aqueous phase. It will be appreciated that for this step, instead of using specifically petroleum ether, it would be possible to employ any solvent capable -of dissolving the fats but incapable of dissolving the adrenal cortical hormone to any great extent.

The solution of the fats is removed from the aqueous residue and the aqueous residue remaining after this separation is extracted with ethylene dichloride. In the specific process we perform three extractions using approximately 14 liters each for the first two times and 7 liters for the last time. The ethylene dichloride here used is highly selective for the adrenal cortical hormone and it removes that hormone almost quantitatively from the aqueous residue and the solution in the ethylene dichloride is in a relatively high state of purity.

We have found that the ethylene dichloride is not a solvent for epinephrln or proteins and that it does not dissolve other objectionable amino compounds that are present in the aqueous phase at this time or which are present in the original mammalian adrenal glands. The ethylene dichloride solution is likewise free from phospholipins and other water soluble impurities present in the aqueous phase treated.

We have found that the total residue in this ethylene dichloride solution of the adrenal cortical hormone has invariably less than 0.1% of the original fresh gland and the inactive impurities which are dissolved in the ethylene dichloride solution consist chiefiy of cholesterol and neutral fat which, if considered objectionable, can be removed by a later step which we will describe. The ethylene dichloride product produced is of such a high grade of purity that it is unnecessary to use separate steps for removing the epinephrln and phospholipins and other impurities and the use of the ethylene dichloride constitutes a very marked simplification of the process for preparing purified extracts containing the adrenal cortical hormone.

We have found that the use of benzol in place of the ethylene dichloride will produce an active extract but that its use requires very complete extracting by the petroleum ether step in the process and the benzol is a solvent to a degree of objectionable phospholipins which we may have to remove by a further purification step. Any solvent highly selective for the adrenal cortical hormone but in which the epinephrln and the other above mentioned objectionable impurities are not soluble would be highly satisfactory. p

The ethylene dichloride solution is separated from the residue and is concentrated to dryness in vacuo at a low temperature, thus removing the solvent. For further purification the dry residue is dissolved in approximately 1 liter of ethyl alcohol and the alcohol solution is mixed with approximately 500 c. c. of petroleum ether and water added sufilcient to make the alcohol concentration approximately 70%.

The petroleum ether layer is then removed and the dilute alcohol phase is extracted with additional quantities of petroleum ether until it is free from the cholesterol. The dilute alcohol solution after separation from' the petroleum ether layer is then concentrated in vacuo to remove the alcohol and the aqueous solution is diluted to the required volume and sodium chloride is added to make the solution isotonic. The addition of sodium chloride at this point salts out aosaua certain impurities by causing the formation of an inactive precipitate which contains considerable colored material which can be easily removed by filtration or centrifugation.

The aqueous solution is then separated from the precipitate and is sterilized by Berkefeld filtration or by any suitable sterilization process.

We have found that a convenient volume of the finished aqueous extract containing the adrenal cortical hormone is one in which 1 c. c. represents 40 grams of the fresh whole adrenal gland. More or less concentrated extracts can beprepared by varying the volume of the isotonic saline used to dissolve the hormone concentrate. We have found that the above extract contains the adrenal cortical hormoneand is substantially free from toxic impurities and is suitable for therapeutic use. The extract containing the active material from 40 grams of fresh whole adrenal gland in each 10. 0. contains less than 1 milligram of solids exclusive of the added sodium chloride for each 1 c. c.

By employing our method it is possible to obtain a new and improved extract of adrenal cortical hormone. The satisfactory qualities of the finished product are due to a great extent to the use of the ethylene dichloride as solvent for the hormone and to the order in which the steps are taken. Ethylene dichloride could be used for the extraction regardless of the order of or of the other steps taken.

Our invention makes possible the reduction of essential operations necessary to preparing the extract suitable for therapeutic use and permits the production at a greatly reduced cost over the methods heretofore employed. The use of the solid carbon dioxide in the acetone at the first of the operation eliminates much objectionable coloring from the final product and prevents the putrefaction of the fresh, whole adrenal glands during the extraction process.

We have described our process in detail for preparing the extract of adrenal cortical hormone from the fresh adrenal glands of animals. The method and the individual steps thereof could be used for preparing the extract of adrenal cortical hormone from other animaltissue and from tissue fiuids and excretion products. It could also be employed to advantage in extracting the adrenal cortical hormone from various aqueous solutions of the hormone however produced and we do not wish to be confined to the specific process used here as an illustration. Having thus described our invention, what we claim as new and desire to secure by Letters Patent is:

1. The method of preparing active extract of adrenal cortical hormone from mammalian adrenal glands, comprising subjecting said glands to the extracting action of acetone, separating the acetone solution, concentrating the acetone solution to remove the acetone and leave an aqueous residue, subjecting the aqueous residue of the concentration of the acetone solution to the extractingaction of petroleum ether, separating the petroleum ether solution from the aqueous residue, subjecting the aqueous residue left after the separation of the petroleum ether solution to the extracting action of ethylene dichloride, separating the ethylene dichloride solution, concentrating the ethylene dichloride solution to remove the ethylene dichloride, dissolving the residue of the concentration of ethylene dichloride solution in ethyl alcohol and adding water to provide the alcohol in a 70% concentration,

subjecting said ethyl alcohol solution to the extracting action of petroleum ether, separating the alcohol solution from the petroleum 'ether solution, concentrating the alcohol solution to remove the alcohol and leave an aqueous residue, adding sodium chloride to the aqueous residue of concentration of the alcohol solution to make. the

aqueous residue isotonic and to precipitate solids,

separating the aqueous residue from the solids, and sterilizing the aqueous residue.

2. The method of preparing active extract of adrenal cortical hormone from mammalian adrenal glands, comprising subjecting said glands to the extracting action of I acetone, separating the acetone'solution, concentrating the acetone solution to remove the acetone and leave an aqueous residue, subjecting the aqueous residue of the concentration of the acetone solution to the extracting actionof petroleum ether, separating the petroleum ether solution from the aqueous residue, subjecting the aqueous residue left after the separation of the petroleum ethersolution to the extracting action of ethylene dichloride, separating the ethylene dichloride solution, and concentrating the ethylene dichloride solution to remove the ethylene dichloride.

3. The method of preparing active extract of adrenal cortical hormones from mammalian adrenal glands, comprising subjecting said glands to the extracting action of a. water soluble solvent capable of dissolving the adrenal cortical hormone, separating said solution, concentrating said solution to remove the solvent and leave an aqueous residue, subjecting the aqueous residue of the concentration of said solution to the extracting action of petroleum ether to remove fat from said aqueous residue, separating the solution of fat from said aqueous residue, subjecting the aqueous residue left after the separation of said solution of fat to the extracting action of ethylene dichloride, separating the solution, and

concentrating said solution ethylene dichloride.

GEORGE F. CARTLAND. MARVIN H. KUIZENGA.

to remove the 

